June 15, 2020 by admin 0 Comments

Dual-crosslinked methylcellulose hydrogels for 3D bioprinting applications

Authors
Ji Youn Shin (a), Yong Ho Yeo (a), Jae Eun Jeong (b), Su A. Park (b), Won Ho Park (a)
Abstract
Thermo-sensitive methylcellulose (MC) hydrogel has been widely used as a scaffold material for biomedical applications. However, due to its poor mechanical properties, the MC-based hydrogel has rarely been employed in 3D bioprinting for tissue engineering scaffolds. In this study, the dual crosslinkable tyramine-modified MC (MC-Tyr) conjugate was prepared via a two-step synthesis, and its hydrogel showed excellent mechanical properties and printability for 3D bioprinting applications. The MC-Tyr conjugate formed a dual-crosslinked hydrogel by modulating the temperature and/or visible light. A combination of reversible physical crosslinking (thermal crosslinking) and irreversible chemical crosslinking (photocrosslinking) was used in this dual crosslinked hydrogel. Also, the photocrosslinking of MC-Tyr solution was facilitated by visible light exposure in the presence of biocompatible photoinitiators (riboflavin, RF and riboflavin 5’-monophophate, RFp). The RF and RFp were used to compare the cytotoxicity and salting-out effect of MC-Tyr hydrogel, as well as the initiation ability, based on the difference in chemical structure. Also, the influence of the printing parameters on the printed MC hydrogel was investigated. Finally, the cell-laden MC-Tyr bioink was successfully extruded into stable 3D hydrogel constructs with high resolution via a dual crosslinking strategy. Furthermore, the MC-Tyr scaffolds showed excellent cell viability and proliferation.

May 29, 2020 by admin 0 Comments

3D-Bioprinted Aptamer-Functionalized Bio-inks for Spatiotemporally Controlled Growth Factor Delivery

Authors
Deepti Rana, Vasileios D. Trikalitis (Contributor), Vincent R. Rangel (Contributor), Ajoy Kumar Kandar (Contributor), Nasim Salehi Nik (Contributor), Jeroen Rouwkema* (Contributor)
Abstract
Introduction Spatiotemporally controlled growth factors delivering systems are crucial for tissue engineering. However, most of the current strategies for growth factors delivery often focuses on the immobilization or coupling of growth factors within the engineered matrices (hydrogel) via various linker proteins or peptides. These systems provide passive release rates and growth factor delivery on demand, but fail to adapt their release rates in accordance with the tissue development. To overcome this limitation, the present study employed nucleic acid based aptamers for achieving spatiotemporally controlled growth factor delivery. Aptamers are affinity ligands selected from DNA/RNA libraries to recognize proteins with high affinity and specificity.1 Aptamer based growth factor delivery systems are able to load/release multiple growth factors on demand with high specificity. In the present study, the authors have 3D-bioprinted aptamer-functionalized bio-inks to evaluate their potential for growth factor sequestering, programmable release and for studying their effect on vascular network formation. Methods The aptamer-functionalized hydrogels were prepared via photo-polymerization of gelatin methacryloyl (GelMA) and acrydite functionalized aptamers having sequence specific for binding to vascular endothelial growth factor (VEGF165). Visible light photoinitiator, tris(2,2′-bipyridyl)dichloro-ruthenium(II) hexahydrate with sodium persulfate was used. The 3D-bioprinting experiments were carried out using Rokit Invivo 3D printer. The viscoelastic properties of the bio-inks were evaluated and compared with control GelMA bio-ink. To study the programmable growth factor release efficiency, VEGF antibody immunostaining was used. For studying the effect of triggered growth factor release on vascular network formation, human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (MSCs) were encapsulated within the bio-inks. Results & Discussion The results obtained from VEGF antibody immunostainings confirmed the sequestration and triggered release of VEGF in response to complementary sequence addition from the 3D bioprinted construct after 5 days of culture. The bioprinted construct showed high cellular viability. The F-Actin/DAPI staining showed cellular sprouting and vascular network formation within the 3D printing aptamer functionalized bio-ink regions. In addition, the endothelial cells showed variations in cellular organization based on the VEGF bound aptamer availability within the bioprinted construct. These observations altogether confirms the bioactivity of VEGF bound aptamers within the printed constructs. Conclusions The present study shows the vasculogenic potential of 3D bioprinted aptamer-functionalized bio-inks via spatiotemporally controlling VEGF availability within the hydrogel system. Acknowledgements: This work is supported by an ERC Consolidator Grant under grant agreement no 724469. References 1. M.R. Battig, et. al., J. Am. Chem. Soc. 134 (2012) 12410-12413.

February 14, 2020 by admin 0 Comments

Photocurable chitosan as bioink for cellularized therapies towards personalized scaffold architecture

Authors
Chiara Tonda-Turo (a) (b), Irene Carmagnola (a) (b), Annalisa Chiappone (b) (c), Zhaoxuan Feng (d), Gianluca Ciardelli (a) (b), Minna Hakkarainen (d), Marco Sangermano (b) (c)
Abstract
Recent progresses in tissue engineering are directed towards the development of technologies able to provide personalized scaffolds recreating the defect shape in a patient specific manner. To achieve this ambitious goal, 3D bioprinting can be combined with a suitable bioink, able to create a physiological milieu for cell growth. In this work, a novel chitosan-based hydrogel was developed combining photocrosslinking and thermo-sensitive properties. Commercial chitosan (CS) was first methacrylated and then mixed with β-glycerol phosphate salt (β-GP) to impart a thermally induced phase transition. The absence of cytotoxic degradation products and the excellent biocompatibility of the developed hydrogel was confirmed through in vitro tests using different cell lines (NIH/3T3, Saos-2, SH-SY5Y). Cellularized 3D structures were obtained though 3D bioprinting technologies confirming the processability of the developed hydrogels and its unique biological properties.

January 1, 2019 by admin 0 Comments

3D Bioprinting of human adipose stem cells (hADSCs) encapsulated hyaluronic acid (HA) based biomimetic double crosslinked hydrogel bioink for cartilage tissue engineering (CTE)

Authors
Parikshit Banerjee
Abstract
Articular cartilage covers the edges of the bones and provides wear resistant load bearing capacity which ultimately supports the flexible joint movement. Therefore, once these articular cartilages get damaged, they limit the free joint movements in patients and cause severe complication. Also, articular cartilage is avascular in nature, which also restricts its ability to repair itself after any damage. To address these issues associated with articular cartilage damage, cartilage tissue engineering (CTE) has been introduced. CTE helps in repairing or regenerating damaged cartilages by using a combined strategy which involves cell, growth factors, and biomaterial scaffolds. Hydrogel with the ability to absorb a large amount of water viewed as an ideal material for cartilage mimetic scaffold owing to the similarity between the hydrogel and native cartilage. Combining stem cell or chondrocytes with hydrogel scaffold is regarded as a promising approach for CTE. This strategy is capable of supporting highly dense cell population, cell attachment, homogeneous cell distribution, and also offer an ideal microenvironment for cell growth and differentiation. Unfortunately, developing hydrogel scaffold with required structural integrity is a major issue that limits the application of hydrogel in CTE. Therefore, to address the problems associated with existing CTE, this thesis aimed to utilize 3D bioprinting to print cartilage constructs by combining adipose-derived stem cells (ADSCs) and hyaluronic acid (HA) based hydrogels. First, to develop a new cartilage extracellular matrix (ECM) mimetic hydrogel system, we synthesized biotinylated-hyaluronic acid (HA-Biotin) and confirmed the successful grafting of biotin with HA trough Fourier Transform Infrared Spectroscopy (FTIR) analysis. Next, HA-Bio hydrogel was prepared and Streptavidin was mixed with this hydrogel to form partially crosslinked HA-based hydrogel through non-covalent bonding between biotin and streptavidin. Addition of streptavidin also supports higher cell attachment due to the presence of cell adhesion sites in streptavidin. After that, partially crosslinked HA-Bio-Streptavidin (HBS) hydrogel was mixed with sodium alginate and subsequently printed using Rokit INVIVO bioprinter. After printing, 3D scaffolds were submerged into CaCl2 solution achieve ionic crosslinking through ion transfer between sodium alginate and CaCl2. Different parameters such as fiber formation, self-supporting ability, printing resolution, and crosslinker concentration were optimized to get desired 3D printed constructs. In vitro cell proliferation and live/dead staining assay were also performed on 3D cell-laden scaffolds. The result showed that partially crosslinking the biotinylated-HA based hydrogel with streptavidin has a significant effect on printability. Morphological analysis of optimal 3D printed scaffold showed clearly visible pores with desired shape and geometry. Favorable cell proliferation and growth was also observed in 3D HBSA based hydrogel scaffolds. These result further confirmed that double crosslinking HA-based hydrogel could be a good choice for 3D bioprinting based tissue engineering.

January 1, 2018 by admin 0 Comments

Design, fabrication and evaluation of a hybrid biomanufacturing system for tissue engineering

Authors
Fengyuan Liu
Abstract
Plasma-assisted Bio-extrusion System (PBS System) is an innovative hybrid bio-manufacturing system to produce complex multi-material and functionally graded scaffolds combining multiple pressure-assisted and screw-assisted printing heads and plasma jets. This approach, which represents a step forward regarding the current state of the art technology in the field of biomanufacturing, enables to design and fabricate more effective scaffolds matching the mechanical and surface characteristics of the surrounding tissue, enabling the incorporation of high number of cells uniformly distributed and the introduction of multiple cell types with positional specificity. The system requires complex control software to manipulate different materials, scaffold designs and processing parameters. This software, developed using MATLAB GUI, is detailed in this paper. It provides high freedom of design allowing the users to create single or multi-material constructs with uniform pore size or pores size gradients by changing the operation parameters, such as geometric parameters, lay-down pattern, filament distance, feed rate and layer thickness. Functionally graded scaffolds can also be designed considering different layer-by-layer coating/surface modification strategies using the multi-jet plasma system. Based on the user definition, G programming codes are generated enabling fully integration and synchronization with the hardware of the PBS system. Examples will be provided describing the design of single, multi-material and functionally graded scaffolds.